Solid formulations such as powders, granules or tablets are widely used in pharmaceutical industry. They typically comprise at least one active ingredient, and may further comprise carriers and other excipients. Solid formulations are also used in other commercial applications, e.g. in the diagnostic area, such as in the manufacture of diagnostic kits. Granules can be used e.g. in capsules, sachets or processed further (e.g. pressed) to tablets. Advantages offered by solid formulations include less storage space, ease of handling, and improved stability. Moreover, tablets or capsules provide the most widely used dosage unit for applying drugs to a patient in a non-invasive manner. A long established practice of preparing solid formulations exists for small molecule active ingredients.
In the meanwhile, immunoglobulins are finding ever-increasing use as active ingredients in therapeutic or diagnostic applications. These applications rely on the antigen binding activity of immunoglobulins.
In comparison to small molecule drugs, immunoglobulins are very large and complex molecules. They carry multiple functional groups and form complex three dimensional structures. The correct folding into a tertiary structure, and, potentially, the assembly of multiple domains or subunits of such three dimensional structures into a quaternary structure are essential for antigen binding. For example, binding of an immunoglobulin variable domain to its antigen depends on the correct formation of the antigen binding site, and thus, on the correct overall folding of the molecule.
Complexity in terms of chemical composition and structure imposes severe limits on methods for preparing solid formulations that comprise biologically active immunoglobulins. The main problem associated with methods for solid formulation of immunoglobulins is protein instability, in particular chemical instability and physical instability.
Chemical instability is caused by changes in the composition of proteins through bond formation or cleavage. Examples of chemical protein instability include deamidation, racemization, hydrolysis, isomerizatin, dehydration, oxidation, beta elimination, glycation, and disulfide exchange/scrambling.
Physical instability affects protein structure. Changes in temperature, shear stress, effects caused by phase interfaces (e.g. liquid/gas), and loss of hydration effects each can result in physical instability of immunoglobulins, such as changes to higher order structure (i.e. aggregation), denaturation or unfolding, adsorption and precipitation. The biological function of macromolecules such as immunoglobulins relies on their native conformation, which is maintained by temperature-sensitive hydrogen bonds or non-covalent interactions between functional groups of the macromolecule. When an immunoglobulin is exposed to increased temperature over a critical level known as the melting temperature (Tm) or the denaturation temperature (Td) it undergoes a sharp structural transition and denatures. Typically this temperature-induced structural transition is irreversible. It is known, for example, that immunoglobulin domains are vulnerable to heat induced unfolding. This in turn leads to exposure of hydrophobic patches which interact to form irreversible aggregates.
It goes without saying that chemical and physical instability interact in compromising biological activity. The resulting loss of activity is incompatible with a pharmaceutical or diagnostic application of such solid immunoglobulin formulations.
All the above effects on physical or chemical stability are favoured by exposure to heat in a liquid state. Moreover, they are favoured by a high interface area between the liquid and gas phase.
It is widely known that proteins can withstand higher temperatures in a dry state than in a liquid state.
Thus, of particular concern for immunoglobulin stability is the combination of heat and a liquid state, in particular under additional shear stress conditions and the presence of large phase interface surfaces. Immunoglobulins that are heated in a liquid state will suffer from chemical modifications, in addition to loosing their proper structure by aggregation and denaturing.
Consequently, strategies have been deployed to avoid temperature induced denaturing. These strategies include a) shorten the time of exposure to high temperature during drying (e.g. spray-drying based on flash evaporation); b) reducing moisture: water content has a great impact on thermal denaturation of proteins being formulated or stored in a powder form. Increase of water content results in a decrease of Td and enthalpy of denaturation and increased protein mobility.
The problems encountered with macromolecular protein therapeutics such as immunoglobulins are not as pronounced in very small peptides. In particular, very small peptides differ in terms of their instability from a chemical, biological and physical point of view. Irreversible conformation changes including aggregation typically are absent in very small peptides. In other words, even if a peptide suffers from conformation changes in the course of a formulation process, it may regain a functional conformation under appropriate conditions and thus regain its activity. For example, solid formulations of insulin are known (e.g. Hosny et al., 2002; J. Pharm. 237(1-2): 71-6). This is in stark contrast to the irreversible changes of macromolecular protein therapeutics which irreversibly loose their activity, and is one reason why much effort has been put in commercializing very small peptides and small molecules instead of proteins in particulate solid dosage forms.
Therefore known methods for preparing solid immunoglobulin formulations avoid the exposure to elevated temperatures in a liquid state and under shear stress. In particular, commonly used methods for solid formulation of immunoglobulins include freeze drying (lyophilization). Freeze drying operates at very low temperatures and thus avoids immunoglobulin instability caused by exposure to heat in a liquid state. However, the solid formulations obtainable by freeze drying typically are not directly suitable for the manufacture of e.g. tablets, capsules or implants. This necessitates complicated and expensive further processing, if such solid dosage forms are to be produced. Therefore, the art attempted to modify and improve freeze drying processes (Leuenberger et al. 2006; Drying Technology 24: 711-719).
Another known method for gentle production of solid state formulations comprising proteins is spray drying, or combinations of freeze-drying and spray drying (e.g. Lee 2000; Pharm. Biotechnol. 13: 135-58; Sollohub and Cal 2010; J. Pharm. Sci. 99(2): 587-97; Vehring 2008; Pharm. Res. 25(5): 99-1022).
Spray drying is based on the principle that a liquid comprising the active agent is sprayed into a hot stream of gas, e.g. air, and vaporised. Droplet size is adjusted (e.g. 20 μm) to maximize surface area for heat transfer and the rate of water evaporation. Solids are formed as moisture quickly leaves the droplets. During this process evaporation has a cooling effect on the droplets. Because of the advantageous ratio of volume to surface area of the droplets, spray dryers can dry a product very quickly compared to other methods of drying. Thus, exposure to heat in a liquid state is reduced to a minimum, and the conversion to a solid state is almost immediate (e.g. in the range of a few seconds). Moreover, the evaporation of the droplets is not associated with shear stress for the active agent.
However, there remains a need for further methods for preparing solid formulations comprising immunoglobulin single variable domains.
The present invention is based on the unexpected finding that a solid formulation comprising, as an active agent, immunoglobulin single variable domains, in particular (camelid) VHH domains, camelized VH domains or humanized VHH domains can be produced by a method combining heat exposure in a liquid state and shear stress, without significant loss of biological activity.